While it has been known for a number of years that mitochondria and non-mitochondrial sources within skeletal muscle produce reactive oxygen species (ROS), the significance of ROS generation has only become clear recently. ROS generation has become implicated in the aging process and numerous disease states, from cancer to heart disease to pathologies related to inflammatory processes. In addition, it has recently become clear that ROS are not only deleterious to some cell functions (inducing apoptosis under extreme conditions) but serve as essential mediators in a number of cell signaling events and have been proposed to modulate important changes in gene expression related to cellular protection from oxidative stress and essential cellular adaptations to exercise. Yet, the factors that modulate ROS metabolism during skeletal muscle contractions and varied conditions of oxygenation remain incompletely understood. In particular, the peculiar behavior of ROS generation during hypoxia, in which higher ROS generation is paradoxically induced despite reduced O2 tensions, has only recently been clearly demonstrated. Finally, while it has been shown that ROS generation in skeletal muscle has significant effects on contractility and muscle function, it remains unclear as to the manner in which generation of ROS in skeletal muscle is dependent on the rate of mitochondrial respiration, work output, intracellular oxygenation, antioxidant buffering, and muscle fiber type. Much of these uncertainties are due to confounding factors related to inhomogeneities within whole muscle models, uncontrolled extracellular environments, variable muscle fiber recruitment patterns, etc. The purpose of this proposal is to use an isolated single mouse skeletal myofiber model, in which the extracellular environment can be precisely controlled and the intracellular environment carefully monitored using non-invasive imaging techniques, to test hypotheses related to delineating the factors regulating ROS generation, the locations of intracellular ROS formation, and ROS modulated gene expression in contracting slow- and fast-twitch fibers under varied oxygenation conditions. In well-controlled experiments in both fiber types with the extracellular PO2 varied during contractions, measurements will be made of mitochondrial respiration and membrane potential, ROS generation and removal, intracellular PO2, contractile function, cytosolic and mitochondrial [Ca2+], numerous intracellular signals related to ROS production, antioxidant protection of cellular integrity, and regulation of gene expression (using QPCR). The strength of this application is in the use of our single muscle fiber model, thereby reducing many of the difficulties related to interpreting results from whole muscle, cell culture, or isolated mitochondria models, and providing data generated from viable, healthy intact myofibers with a normal physiological intracellular environment. This application provides a singular opportunity to study the factors that regulate ROS metabolism in skeletal muscle and thereby has significant implications for understanding and potentially improving human health. PUBLIC HEALTH RELEVANCE. It has only recently become clear that the production of reactive oxygen species during exercise and during low oxygenation conditions has a significant impact on muscle function and adaptation. It is the goal of this proposed research to elucidate the regulatory factors that influence reactive oxygen species production during exercise and low oxygen conditions and how these changes affect muscle performance and the adaptive response to exercise.